Comparison of long-read and short-read bacterial DNA sequencing

Abstract

This study aimed to compare long-read (Oxford Nanopore) and short-read (Illumina) sequencing technologies for characterizing the diversity and composition of in vitro oral biofilms. An oral biofilm model was established using hydroxyapatite discs to mimic the tooth surface. Saliva samples from six healthy participants were pooled and used to inoculate the discs, which underwent aerobic and anaerobic incubation phases. Biofilm formation was assessed using confocal microscopy with LIVE/DEAD staining, revealing heterogeneous growth patterns with coverage ranging from 17% to 47%. DNA extraction was carried out using the GenElute Bacterial Genomic DNA Kit, with yields showing significant variations across experiments (0-5 ng/μL). Interestingly, gel electrophoresis showed no difference in DNA fragment lengths between samples prepared for short-read and long-read sequencing. The experiment also highlighted the potential benefits of CO2-rich environments for early colonizer growth, particularly Streptococcus species. While the biofilm model was successfully established, the results underline the need for protocol optimization, particularly in DNA extraction and biofilm cultivation. This research provides insights into the complexity of oral microbiome analysis and sets the stage for future comparative studies on advanced sequencing technologies in oral microbiome. The findings also emphasize the importance of refining the in vitro experimental protocol, from biofilm cultivation to DNA sequencing and data analysis.